In the United States, urogenital infections with Chlamydia trachomatis are the leading sexually transmitted disease with an estimated 10 million new cases reported each year. The urogenital infections result in involuntary infertility in 100-200,000 women each year. It is estimated that equal or greater numbers of these infections occur in European countries. In addition, ocular infection with C. trachomatis results in blinding trachoma which afflicts approximately 500 million individuals from developing countries. Improved diagnostic methodologies and vaccines or immunoprophylactic preventative measures are needed to manage and control the disease.
Chlamydia trachomatis isolates occur as 15 distinct serovars. Based on serological relatedness, these 15 serovars have been divided into three serogroups; B serogroup (serovars B, Ba, D, E, L1 and L2), intermediate serogroup (serovars F, G, K and L3), and C serogroup, (serovars A, C, H, I and J). The antigen that confers serovar and serogroup-specificities to chlamydia is the major outer membrane protein (MOMP), and protective immunity developed during chlamydial ocular infection is thought to be directed at serovar and serogroup MOMP determinants.
The genes encoding the MOMP of the C. trachomatis serovars A, B, C, L1 and L2 have been cloned and sequenced (Pickett et al., FEMS Microbiol. Lett. 42:185-190 (1987); Stephen et al., J. Bacteriol. 168:1277-1282 (1986); and Stephens et al., J. Bacteriol 169:3879-3885 (1987); Baehr et al., Proc. Natl. Acad. Sci. USA, 85:4000-4004 (1988)). Comparative analysis of their amino acid sequences show the MOMP genes to be highly conserved structures that contain four evenly spaced domains whose sequences vary among the different serovars. The locations of these nucleotide and amino acid sequence variable domains (VDs) are:
VDIxe2x80x94nucleotides 256-315 and residues 64-83;
VDIIxe2x80x94nucleotides 481-546 and residues 139-160;
VDIIIxe2x80x94nucleotides 736-777 and residues 224-237;
VDIV xe2x80x94nucleotides 928-1017 and residues 288-317.
Epitope mapping has shown that three of the four VD domains (I, II and IV) contain contiguous antigenic determinants which elicit the formation of serovar, subspecies (determinants common to three or more serovars within a serogroup), serogroup or species-specific antibodies.
Variable domains I and II, which demonstrate the greatest amount of inter serogroup sequence variation, are the locations of the serovar-specific determinants. Variable domain IV is the largest of the MOMP VDs and is located near the C-terminus of the protein. VDIV is the location of subspecies and serogroup antigenic determinants, as well as a highly conserved species-specific antigenic determinant. Antigenic determinants have not been mapped to VDIII, the smallest and least variable domain of the MOMP genes.
Variable domains I, II and IV of the MOMP of C. trachomatis protrude from the cell surface towards the external environment as demonstrated by their susceptibility to cleavage by trypsin, and their accessibility to antibody binding. Trypsin cleavage in both VDII and VDIV, but not within VDIV alone, decreases chlamydial attachment to HeLa cells suggesting that these domains, or conformational MOMP structures that are dependent on the integrity of these domains, may function as a chlamydial ligand.
Based on the immunological and biological relationships among MOMP VD structure and function, and the fact that the MOMP VDs appear to be the major variable sequences between serovars, the present inventors sought to identify the nucleotide and amino acid sequences of the MOMP VDs I-IV of C. trachomatis serovars Ba, D, E, F, G, H, I, J, K and L3, and to confirm those of serovars B, A, C, L1 and L2.
The amino acid sequences of the major outer membrane protein genes from Chlamydia trachomatis serovars A, B, C, L1 and L2 have been shown to be conserved with the exception of four variable domains in which major neutralizing and serotyping antigenic determinants have been located. Using oligonucleotide primer extension sequencing of MOMP mRNA, the nucleotide sequences of the four MOMP VDs of the remaining 10 C. trachomatis serovars were determined by the present inventors and the amino acid sequences deduced therefrom. The sequencing technique was rapid and required minimal amounts of total RNA, i.e., only 35 xcexcg of RNA from chlamydial infected HeLa cells was needed to sequence all 4 variable domains for a given serovar. Use of the disclosed technique also circumvented the more standard and laborious approach of molecular cloning and direct DNA sequencing which had been employed in the past to determine the sequences of the MOMP gene to serovars A, B, C, L1 and L2.
As noted above, the nucleotide sequences were employed to deduce the corresponding amino acid sequences. Comparative analysis of the amino acid sequence homology of the four variable domains permitted the present inventors to separate the fifteen serovars into three serogroups:
Group 1xe2x80x94serovars B, Ba, D, E, L1 and L2;
Group 2xe2x80x94serovars G and F;
Group 3xe2x80x94serovars A, C, H, I, J, K and L3.
The four variable domains were also analyzed for immunogenicity based on the hydrophilicity and charge values of each domain (Baehr et al., supra). The MOMP VDs with the greatest total hydrophilicity and charge values were found to be the locations of the antigenic determinants recognized by MOMP specific monoclonal antibodies (Stephens et al., supra; Baehr et al., supra).
The nucleotide, amino acid sequences and hydrophilicity/charge value analyses are advantageous because they will assist in the selection of appropriate MOMP antigenic determinants to be used in the construction of synthetic peptides, subunits or recombinant chlamydial vaccines. Examples of such recombinant vaccines include infectious enteric vectors (avirulent Salmonella typhimurium and/or Sabin Type I polio virus) expressing MOMP variable domain sequences as antigenic chimeras or hybrid molecules.
The present invention will allow the production of reagents and methodologies applicable in the development of new diagnostic tests for C. trachomatis infections and serological tests for serotyping. Specifically, species or serotype specific oligopeptides conjugated to an array of reporter or detector groups could be used in various conventional non-immunologic assays including standard hybridization techniques.
Likewise, reagents for use in immunologic or serologic tests could be prepared based on the sequences presently disclosed; the conserved VD-IV peptide sequence TTLNTTIAG would permit species specific identification, while the non-conserved sequences would enable one to make sero-type specific identification. It is contemplated by the present inventors that such reagents would be suitable for use in standard immunologic procedures such as ELISA and radioimmunoassay techniques.
The invention will be more fully described in the detailed description and drawings which follow.